Process for obtaining proteohormones with albumen character



Patented Oct. 29, 1940 rmenss non Hammer Erich Babald, w

amber} and rrluf Johannessohn, Mannheim Feudenheim, oermany,assignors-to Bare Chemicals, Inc., Nepera N. E, a corporation of-NewYork 1 No Drawing. Application June 25, 1938, Serial No. 215,814. InGermany June 28, 1937 8 Claims. (01. 260-112) The present inventionrelates to processes in-' volving the preparation and purification orproteo hormones and the products of such processes. Another object ofthe invention is to prepare new and useful compositions and compounds ofhormones with albumen character. Further objects will hereinafterappear.

We have discovered thatproteo hormones can be precipitated from theiraqueous solutions or m aqueous organic solvents by means of alkaloidswhich are dimcultly soluble or insoluble in water. This fact is the moresurprising as such alkaloids do not belong to the albumen preclpitants.The present invention therefore-relates to a process for obtainingproteo hormones with albumen character characterized by treatingsolutions or liquids containing such hormones, especially body liquidssuch as urine or serum, with alkaloids which are diflicultly soluble orinsoluble in water. Such hormones are for example the prolactin, theinsulin, the gonadotropic hormone and the thyreotropic hormone. Suitablealkaloids are for example those of the purine group, e. g. theobromine,brucine, but particularly the alkaloids of the quinine group, such asquinine and quinidine. However, it is not necessary, to add the alkaloidbases as such, but the new compounds may also be produced byprecipitation from alkaloid salts, which may be mixed with the hormonecontaining fluid, with stronger bases such as caustic soda, aqueousammonia, and so'forth. Also difflcultly soluble alkaloid salts, such asbasic quinine citrate, basic quinine tartrate, quinine oxalate, etc.,may be used.

In this manner addition compounds are obtained which are diflicultlysoluble in water and which can be separated from theirmotherliquors inthe usual manner, for example by filtration or centrifuging. From thesecompounds the 40-hormones can easily be liberated by treating them witha solvent for the one or the other of the constituents. For example thehormone constituent can be separated by means of a suitable aqueoussolution and the alkaloid constituent is left undissolved, or theaddition compounds may be decomposed by adding organic solvents, forexample absolute alcohol, whereby only the alkaloid constituent isdissolved. The hormone products thus obtained have a high degree ofPurity.

' Ezeamples (1) 500 g. of hypophysis anterior lobes of oxen are finelyground and then pressedin a 'press.

The residue from pressing is" thrice itriturated basic salt, ammoniumsulphate and the like.

each time with 300 cc. of 0.01 normal caustic soda lye and pressed altereach tritutration. Thus altogether 1200 cc. of liquid are obtained,which is adjusted to pH=7. 12 g. of quinidine dissolved in a smallquantity of alcohol are added 5 to this liquid. The iormed precipitateis filtered by suction, sucking it as dry as possible, and thenextracted with 96% alcohol, more than 10 g. of-

quinidine going into solution. The residue is dissolved in alcoholhaving a pH of about 9' 10 to 9.5. The remaining small quantity of qulnidine is filtered on and the liquid brought to a pH of about 5.5. So muchabsolute alcohol is now added, that the alcohol content of the liquidamounts to to The precipitate which is 15 formed by allowing tov standrepresents a prolactine concentrate which already shows the pigeon unitin 10 mg. (Under a pigeon unit is to be understood the quantityof agentwhich after five intramuscular injections within five days in two 30 offour pigeons produces, on the sixth day an enlargement of the crop whichis just perceptible.) For further purification the concentrate isdissolved in water by adding a few drops of caustic soda lye, so that asolution of about 1% is pro- 25 v duced. This solution is then mixedwith hydrochloric acid until a pH of about 4 is obtained. The mainquantity of the prolactin precipitates at this occasion,whereas-by-products e. g. the

gonadotr opic and thyreotropic' hormoneremain so j in solution. Duringtheprecipitation it is ad visable to assist the flocculation by adding.rock

The m'oth'er liquor can be mixed once more with hydrochloric'acid untila pH of about5L5' g1 is obtained; The flocculation'occurrin'g at thisoccasion consists again of prolactin which is added to'themai'nlquantity. It is then dried in the usual manner with acetone. Theprolactin thus obtained has the pigeon unit in 4 to 6 mg. 1 4o i Theconcentrate the pigeon unit of which is 10 mg. may also be furtherpurified by shaking .itwithfcalcium carbonate, the inactive compon--,en'tsf be ng; then absorbed by the chalk. The

filtra te" produces after a "precipitation in the 5 abovedesed'lmanner'at' a pH of about 5.5 a

prolactinwith the pigeon unit in 3 to 5 mg.

(2') '501g of-:dry powdered pig hypophysis are ground in a ball millwith500 cc. of water of a pH oi about 4.5 or with a suitable buffersolution, 50

. the temperature being kept as low as possible. Itis then filtered anda second extraction carried out. Thefl'aqueous extract thus obtained (1liter) :isfthen shaken with 30 g. of quinidine.

Thereby the active substance combines with the u quinidine. Alterintensively'sucking of! the residue is exhaustively extracted withabsolute alcohol. The residue is then dissolved in the smallest possiblequantity of water of a pH of about 4:5 and again precipitated withabsolute alcohol. Thus concentrates of the gonadotropic hormone areobtained which contain the Evans unit in 1 to 2 mg.

(3) 1 liter of serum from the blood of pregnant mares is brought to a pHof about 4.5. Any flocculations are filtered on and the filtrate isshaken with 8 g. of quinine. The mixture is then again filtered and theresidue extracted with absolute alcohol and subjected to furthertreatment as described in Example 2.

Various changes may be made in the details disclosed in the foregoingspecification without departing from the invention or sacrificing theadvantages thereof.

We do not limit ourselves to the specifically mentioned times,temperature, quantities, chemicals or steps of procedure as these aregiven simply to clearly describe our invention as set forth in ourspecification and claims, and they may be varied without going beyondthe scope of our invention.

We claim:

1. Process of obtaining proteo hormones in treating a liquid obtained byrepeated lixiviating and subsequent pressing of hypophysis anteriorlobes with an in water diflicultly soluble and insoluble alkaloid baseat a pH=7, filtering the .arising difllcultly soluble addition compoundby suction, extracting the latter with absolute alcohol, dissolving theresidue in alcohol having a pH of about 9 to 915, bringing the filteredliquid to a pH of about 5.5 and precipitatingthe hormone hydrochloricacid until a by adding so much absolute alcohol until the alcoholcontent of to to I v 2. Process of obtaining proteo hormones contheliquid amounts sisting in treating a pressed juice of hypophysisanterior lobes according to claim 1, dissolving 5 precipitated hormonefor further purification 15 with 'calcium carbonate, mixing the filtratewith pH of about 5.5 is obtained and -drying the' precipitated prolactinwith acetone.

4. Process of obtaining 'proteo hormones by m treating a pressed juiceof pig hypophysis with an in water difiicultly soluble and insolublealkaloid base. sucking oil! the dimcultly soluble addition compound andisolating thence the gonadotropic hormone by hol.

the gonadotropic hormone and'quinine base.

8. A diificultly soluble addition compound of a member. oi the groupconsisting of prolactin hormone and gonadotropic hormonewith a member 3501 the group consisting of quinidine base and quinine base.

@ERI-CH RABALD? JOHANNESSOHN.

precipitation of the prolactin l0 extraction with absolute alco-

